Alzheimer's disease, heart disease, stroke, Parkinson's disease, and diabetes are leadings causes of death in the United States and therefore have a large impact on human health and quality of life. Numerous studies, both in vitro and in vivo have implicated c-jun-N-terminal kinase (JNK) as being extremely important for cell death and survival in key cell types affecting these diseases such as neurons, cardiomyocytes, and -islets. Much of this cell death occurs through oxidative stress mechanisms and mitochondrial dysfunction. Many ATP-competitive kinase inhibitors have been discovered, but few, if any substrate competitive have been advanced. The goal of this work therefore is to develop high-throughput biochemical assays that can discover selective substrate competitive (non-ATP competitive) inhibitors of c-jun-N-terminal Kinase (JNK) and phosphorylation state selective inhibitors. This will take kinase assay development and drug discovery in a bold new direction. In addition, high-throughput cell-based assays and functional assays will be established to understand the function of these inhibitors in preventing mitochondrial dysfunction and cell death. Developing assays to discover compounds that are substrate competitive inhibitors of JNK provides a novel mechanism to design highly selective JNK inhibitors that likely may not have potential side effects associated with strict ATP competitive kinase inhibitors. To accomplish this we will: 1) Develop a fluorescence polarization (FP) assay for discovering substrate and bidentate competitive selective inhibitors, and fluorescence resonance energy transfer (FRET) assays for activation (phosphorylation) state selective inhibitors. 2) Develop an immunocytochemical cell-based (In-Cell Western) to detect inhibition of c-Jun phosphorylation in SHSY5Y cells, and 3) Establish functional cell-based assays in SHSY5Y cells to monitor reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cell death. Collectively, these three aims will provide the backbone for a novel drug screening platform that will allow for unique compound discovery and neuronal cell-based functional assays.